Roots are an important organ of higher plants. Their main functions are anchoring of the plant in the soil and uptake of water and nutrients (N-nutrition, minerals, etc.). Thus, root growth has a direct or indirect influence on growth and yield of aerial organs, particularly under conditions of nutrient limitation. Roots are also relevant for the production of secondary plant products, such as defense compounds and plant hormones.
Seeds are the reproduction unit of higher plants. Plant seeds contain reserve compounds to ensure nutrition of the embryo after germination. These storage organs contribute significantly to human nutrition as well as cattle feeding. Seeds consist of three major parts, namely the embryo, the endosperm and the seed coat. Reserve compounds are deposited in the storage organ which is either the endosperm (resulting form double fertilisation; e.g. in all cereals), the so-called perisperm (derived from the nucellus tissue) or the cotyledons (e.g. bean varieties). Storage compounds are lipids (oil seed rape), proteins (e.g. in the aleuron of cereals) or carbohydrates (starch, oligosaccharides like raffinose).
Starch is the storage compound in the seeds of cereals. The most important species are maize (yearly production ca. 570 mio t; according to FAO 1995), rice (540 mio t p.a.) and wheat (530 mio t p.a.). Protein rich seeds are found in different kinds of beans (Phaseolus spec., Vicia faba, Vigna spec.; ca. 20 mio t p.a.), pea (Pisum sativum; 14 mio t p.a.) and soybean (Glycine max. 136 mio t p.a.). Soybean seeds are also an important source of lipids. Lipid rich seeds are as well those of different Brassica species (app. 30 mio t p.a.), cotton, oriental sesame, flax, poppy, castor bean, sunflower, peanut, coconut, oilpalm and some other plants of less economic importance.
After fertilization, the developing seed becomes a sink organ that attracts nutritional compounds from source organs of the plant and uses them to produce the reserve compounds in the storage organ. Increases in seed size and weight, are desirable for many different crop species. In addition to increased starch, protein and lipid reserves and hence enhanced nutrition upon ingestion, increases in seed size and/or weight and cotyledon size and/or weight are correlated with faster growth upon germination (early vigor) and enhanced stress tolerance. Cytokinins are an important factor in determining sink strength. The common concept predicts that cytokinins are a positive regulator of sink strength.
Numerous reports ascribe a stimulatory or inhibitory function to cytokinins in different developmental processes such as root growth and branching, control of apical dominance in the shoot, chloroplast development, and leaf senescence (Mok M. C. (1994) in Cytokines: Chemistry, Activity and Function, eds., Mok, D. W. S. & Mok, M. C. (CRC Boca Raton, Fla.), pp. 155-166). Conclusions about the biological functions of cytokinins have mainly been derived from studies on the consequences of exogenous cytokinin application or endogenously enhanced cytokinin levels (Klee, H. J. & Lanehon, M. B. (1995) in Plant Hormones: Physiology, Biochemisry and Molecular Biology, ed. Davies, P. J. (Kluwer, Dordrdrocht, the Netherlands), pp. 340-353, Smulling, T., Rupp, H. M. Frank, M& Schafer, S. (1999) in Advances in Regulation of Plant Growth and Development, eds. Surnad, M. Pac P. & Beck, E. (Peres, Prague), pp. 85-96). Up to now, it has not been possible to address the reverse question: what are the consequences for plant growth and development if the endogenous cytokinin concentration is decreased? Plants with a reduced cytokinin content are expected to yield more precise information about processes cytokinins limit and, therefore, might regulate. Unlike other plant hormones such as abscisic acid, gibberellins, and ethylene, no cytokinin biosynthetic mutants have been isolated (Hooykens, P. J. J., Hall, M. A. & Libbeuga, K. R., eds. (1999) Biochemistry and Molecular Biology of Plant Hormones (Elsevier, Amsterdam).
The catabolic enzyme cytokinin oxidase (CKX) plays a principal role in controlling cytokinin levels in plant tissues. CKX activity has been found in a great number of higher plants and in different plant tissues. The enzyme is a FAD-containing oxidoreductase that catalyzes the degradation of cytokinins bearing unsaturated isoprenoid side chains. The free bases iP and Z, and their respective ribosides are the preferred substrates. The reaction products of iP catabolism are adenine and the unsaturated aldehyde 3-methyl-2-butonal (Armstrong, D. J. (1994) in Cytokinins: Chemistry, Activity and Functions, eds. Mok. D. W. S & Mok, M. C. (CRC Boca Raton, Fla.), pp. 139-154). Recently, a cytokinin oxidase gene from Zea mays has been isolated (Morris, R. O., Bilyeu, K. D., Laskey, J. G. & Cherich, N. N. (1999) Biochem. Biophys. Res. Commun. 255, 328-333, Houba-Heria, N., Pethe, C. d'Alayer, J & Lelouc, M. (1999) Plant J. 17:615-626). The manipulation of CKX gene expression could partially overcome the lack of cytokinin biosynthetic mutants and can be used as a powerful tool to study the relevance of iP- and Z-type cytokinins during the whole life cycle of higher plants.
The present invention overcomes problems related to containment of auxin effects, maintenance of root outgrowth, and promotion of increased seed, embryo, and cotyledon size and/or weight through reduction of endogenous cytokinin concentration.
WO99/06571 discloses the first cloning of a cytokinin oxidase, and plants transformed with the maize cytokinin oxidase were presented. In the same document the idea is proposed to modulate cytokinin levels in plant cells by modifying cytokinin oxidase levels for altering pathogen resistance or plant growth properties (like increased grain production or desired secondary growth characteristics). A practically worked out example is found in US2003163847, showing a method for producing male sterile plants by increasing cytokinin oxidase expression under control of a pollen or anther specific promoter, such that pollen formation or male organ development was inhibited. Increasing cytokinin levels back to normal restored male fertility. Plants overexpressing CKX genes under control of a constitutive promoter had various desirable characteristics, including enhanced root growth and altered root geotropism, increased leaf thickness, parthenocarpy, improved standability of seedlings, increased branching, improved lodging resistance, whereas downregulating cytokinin oxidase expression resulted in plants having delayed leaf senescence, increased shoot meristems, increased vessel size (WO01/96580). An attempt to modify seed yield using cytokinin oxidase is presented in WO00/63401 by Habben et al. This disclosure aims at improving seed size, decreasing tip kernel abortion, increasing seed set during unfavourable environmental conditions and stability of yield by increasing cytokinin levels in the developing seed. The two approaches proposed are to either inhibit or abolish cytokinin oxidase activity with antagonists or through antisense molecules, or to increase the synthesis of cytokinin. The opposite strategy is used in WO03/050287 for increasing seed yield. Schmülling et al. demonstrated that increased seed size/weight, increased embryo size/weight or increased cotyledon size/weight is obtained in tobacco or Arabidopsis by overexpressing a CKX gene under control of a constitutive promoter. Also the use of seed specific, embryo specific or cotyledon specific promoters is contemplated for reaching this goal.
It has now surprisingly been found that heterologous expression of a nucleic acid molecule encoding a cytokinin oxidase (hereafter abbreviated as CKX) in the shoot of a plant gives rise to plants having modified growth characteristics, especially increased yield, in particular increased seed yield. The present invention therefore provides a method for increasing seed yield of a plant, comprising introducing and expressing in said plant an isolated nucleic acid molecule encoding a cytokinin oxidase/dehydrogenase, a homologue, a derivative or an active fragment thereof, characterised in that said expression is primarily obtained in the shoot of said plant.